Brain tumors are the most common cause of oncological death in American children, and medulloblastoma is the most common malignant childhood brain tumor, with over 500 cases diagnosed each year. While therapy for standard-risk patients has resulted in improved outcomes, high-risk patients with Myc oncogene overexpression still do poorly. In addition, there remains significant therapy-related morbidity, particularly in very young patients. Thus, there is a critical need for more effective therapies to combat high Myc expressing medulloblastoma. Using integrated genomic analysis we have recently identified the WEE1 kinase as a potential therapeutic target in medulloblastoma. We demonstrated that WEE1 is over expressed in medulloblastoma patient samples and that inhibition of WEE1 suppresses medulloblastoma cell growth in vitro and in vivo. In particular Myc overexpressing cells are more sensitive to WEE1 inhibition. However how WEE1 enhances medulloblastoma tumorigenesis is unknown. Further whether Myc driven medulloblastoma can be treated with current clinical inhibitors of WEE1 is not known. Thus the objectives of this proposal are to determine the mechanism of WEE1 activity in medulloblastoma and to provide pre-clinical validation of WEE1 inhibition as a therapeutic approach in Myc driven medulloblastoma. We hypothesize that WEE1 enhances medulloblastoma cell survival by protecting cells from Myc oncogene induced replicative stress and promoting DNA damage repair in response to DNA replication targeted chemotherapeutics. To address the hypothesis the studies in aim one will determine the role of WEE1 in medulloblastoma tumorigenesis by examining the impact of WEE1 expression in Myc expressing medulloblastoma cells, examining how WEE1 co-operates with Myc in transforming human neural stem cells and evaluating the synthetic lethal interaction of Myc expression with WEE1 inhibition. Aim two is designed to establish the therapeutic efficacy and tolerability of WEE1 inhibition in vivo using a specific WEE1 inhibitor, MK 1775, that is currently in clinical development. Aim three will identify determinants that mediate synthetic lethality with WEE1 inhibition in medulloblastoma using genome wide RNAi technology and kinase inhibitor high throughput systems. The proposed studies will define how WEE1 regulates medulloblastoma tumorigenesis and establish WEE1 as a novel therapeutic target in medulloblastoma by providing the scientific rationale and preclinical data required for early phase clinical studies. Completion of these studies is expected to impact medulloblastoma therapy by resulting in novel therapeutic strategies incorporating WEE1 inhibition.